Throughout the HIV life cycle, viral-host complexes play an integral role in the biology of the virus. Assemblies range from binary protein-protein and protein-nucleic acid complexes to much larger multicomponent complexes. High-resolution structures of these complexes offer the potential to provide templates for intervention strategies in the treatment of AIDS, but structures have so far been solved in only a few cases. Obtaining such structural information has been limited in part because several of the HIV proteins are poorly behaved biochemically and often are unstructured in isolation. These characteristics may reflect an inherent property of the proteins to adopt different conformations when bound transiently to different cellular partners throughout the viral life cycle. This project, called the HARC Center (HIV Accessory and Regulatory Complexes), focuses on five key HIV proteins that perform important regulatory and accessory functions - Integrase (IN), Tat, Rev, Vif, and Nef- and their interacting viral and cellular protein partners. Our primary aim is to achieve a comprehensive structural picture of HIV-host cell interactions formed during early phases of the viral life cycle, particularly those that regulate gene expression, cell signaling, or cell biology. Several of these viral and host proteins undergo significant conformational changes when complexed to their nucleic acid or protein partners, and understanding the molecular details of this dynamic behavior is an important structural goal. Our focus on regulatory complexes will provide new insights into the biology and pathogenesis of HIV and define new therapeutic targets to combat HIV infection. The HARC Center will harness protein expression and co-expression strategies to characterize functional complexes using a comprehensive battery of structural methods, including mass spectrometry, x- ray crystallography, NMR, and cryo-EM. To overcome the obstacles that have limited progress to date, we propose substantial efforts for technological development. Essential new methods will be explored to optimize expression-to-crystallization, analyze protein dynamics using crystallographic and NMR data, and determine structures of small and heterogeneous complexes by cryo-EM. Strong outreach and collaborative components will draw investigators from outside the Center, particularly those with expertise in HIV mechanisms, protein biochemistry, and virology. OVERALL